Squamous cell carcinoma (SCC) of the oral cavity is one of the most common cancers in the world. The long-term survival rate for this cancer is only about 50%. Therefore, new approaches for the prevention and therapy of human oral cancer are needed. RNA interference (RNAi) inhibits gene expression by degrading specific mRNAs. Previous studies showed that siRNA duplexes could be taken up efficiently by the mucosal tissues of mammals, resulting in a decrease in target gene expression. In this proposed research, our focus is to establish innovative local siRNA delivery approaches by which the mRNA and protein levels of genes activated in oral cancer can be effectively knocked down in the mouse model of oral cancer previously generated in our laboratory. Because both cyclooxygenase 2 (Cox-2) and cyclin D1 proteins are overexpressed in human oral cancer and inhibition of their expression suppresses the proliferation of human oral cavity cancer cells, they are potentially excellent targets for siRNA knockdown to test our approaches. The information gained from this proposed study will be very useful in the design of novel and innovative siRNA approaches for the prevention and therapy of human oral cancer. Our Specific Aims for this proposed study are: 1) To design effective routes for siRNA delivery into the epithelial cells of the mouse tongue and to measure the gene silencing effects of applied siRNAs. The data from these experiments will allow us to determine the doses of siRNA, and the frequencies of the treatments required for the future siRNA studies in the human oral cancer mouse model. 2) To design siRNAs for mouse Cox-2 and cyclin D1 and to test their specificity and potential off-target effects by using cultured primary mouse keratinocytes. The results from these experiments will allow us to determine the most appropriate siRNAs to be used in Specific Aim 3 to test the potential preventive and therapeutic effects of these siRNAs in oral cavity cancer. 3) To investigate the effects of the siRNAs designed in Specific Aim 2 on the development of mouse oral cavity tumors by using the murine oral cavity and esophageal cancer model previously generated in our laboratory. This model demonstrates similarities to human oral cavity carcinogenesis in terms of its morphological, histopathological, and molecular characteristics. We will test our hypothesis that locally delivered, specific Cox-2 and cyclin D1 siRNAs will reduce the levels of endogenous Cox-2 and cyclin D1 proteins in the tumors, and will possibly inhibit oral (tongue) carcinogenesis induced by 4-NQO. Even if we see no differences in tumor incidence in the 4-NQO treated mice as a result of the Cox-2 or cyclin D1 siRNA intervention, if we can convincingly show that we can reduce Cox-2 and/or cyclin D1 protein levels with local siRNA delivery to mouse tongues, Specific Aim 3 will be considered to be successful. The results from all of our proposed experiments should lead to the development of very effective delivery mechanisms for local delivery of siRNA therapies to humans with oral cancer or at risk for oral cancer (e.g. patients with leukoplakia). PUBLIC HEALTH RELEVANCE: Human oral cavity cancer is one of the most common cancers in the world, with a low long-term survival rate. The proposed study will explore and develop a novel approach for the prevention and therapy of human oral cancer by using the local delivery of small interfering RNAs (siRNAs) as a type of drug to decrease the expression of some proteins involved in the development of human oral cavity cancer.